Plasminogen activators are a class of serine proteases that convert plasminogen into plasmin. Plasmin degrades the fibrin matrix of blood clots, thereby restoring the hemodynamic condition of an open vascular system after an internal vascular accident has produced thrombosis or thromboembolism. Vascular disease states involving partial or total blockage of blood vessels which are amenable to treatment with plasminogen activators include stroke, pulmonary embolism, myocardial infarction, as well as deep vein and peripheral artery obstructions.
There are two immunologically distinct types of plasminogen activators found in human plasma and other body fluids--the urokinase-type plasminogen activator (u-PA; M.sub.r, 55,000) and the tissue-type plasminogen activator (t-PA; M.sub.r, 68,000). The activity of the tissue-type plasminogen activator is potentiated by fibrin. The enzyme acts at the site of a thrombus and demonstrates a higher affinity for fibrin than does the urokinase-type plasminogen activator (Haylaeris et al., J. Biol. Chem., 257, 2912, 1982). Therefore, the tissue-type plasminogen activator is considered to be the physiologically relevant thrombolytic agent.
Both activators, u-PA and t-PA, share the following common features: 1) they are synthesized as single chain proenzymes which can be cleaved by plasmin or trypsin, without disrupting their disulfide linked two-chain molecular structure. Upon reduction, each plasminogen activator breaks down into a heavy and a light chain (M.sub.r 33,000 for u-PA; M.sub.r 35,000 for t-PA); 2) both enzymes are serine proteases which can be inactivated by serine-specific reagents such as diisopropyl fluorophosphate; and 3) both enzymes contain a triple disulfide-linked sequence of amino acids which form a loop or kringle in the molecule. Urokinase plasminogen activator has a single kringle. Tissue plasminogen activator has two kringles connected by a hexapeptide linker sequence. These kringles are believed to be responsible for binding of the enzymes to fibrin (Thorsen, Biochem. Biophys. Acta., 393, 55, 1975).
The DNA sequence analysis and the amino acid sequence of t-PA is disclosed by Pennica et al., Nature, 301, 214, (1983), Ny et al., Proc. Natl. Acad. Sci. U.S.A. 81 5355 (1984) and European Patent Application 93,619 to Genentech Inc. The DNA sequence analysis and the amino acid sequence of u-PA is disclosed in European Patent Application 92,182 to Genentech Inc. and urokinase cDNA is discussed by Verde et al., Proc. Natl. Acad. Sci. U.S.A. 81 4727 (1984).